All-trans-retinoic acid (ATRA) is the standard therapy for acute promyelocytic leukemia (APL). Clinically, patients respond to ATRA through complete remissions associated with myeloid maturation of promyelocytic leukemic blast cells. These remissions are transient and relapsed APL is often ATRA-resistant. The mechanisms of ATRA resistance are largely unknown. In this study, we used stable isotope coding quatitative proteomics approach to determine differences in the protein expression levels in ATRA-sensitive NB4 APL cells versus NB4.R1 (a de novo ATRA-resistance derivative of NB4 cell line), after exposing to ATRA. While NB4.R1 cells were grown in normal RPMI medium, NB4 cells were grown in RPMI medium in which normal leucin was replaiced with deuterium labeled leucin. Proteins from each cells were mixed 1:1 ratio and then separated by 1-dimentional SDS-PAGE. Selected bands were excised and trypsin digested and later separated and identified using a high performance quadrupole time-of-flight (QqTOF) mass spectrometer with a mass range of m/z 5-40,000. Proteomics profile revealed that several mitochondrial proteins, including the radical oxygen species scavengers peroxiredoxins (PRDXs), ADP-ATP translocase (ANT-2), various proteins of the mitochondrial electron-transport chain and TNF receptor-associated protein 1 (TRAP-1), were differentially regulated in NB4 vs. NB4.R1 cells. Western blot analysis showed that both mitochondrial PRDX-3 and the cytoplasm PRDX-1 proteins decreased in a time-dependent manner after ATRA treatment in NB4 cells but did not change in NB4.R1. Suppression of PRDX-1 expression by small interference RNA (siRNA) had not apparent effects on differentiation of NB4 cells, detected by CD11b expression. We are currently investigating whether PRDXs are involved in ATRA-induced apoptosis and whether suppression of PRDX-3 expression by siRNA attenuates the resistance of NB4.R1 cells to the ATRA treatment. Overall our results indicates that PRDXs and other mitochondrial-related proteins could be involved in development of ATRA-resistance, and in the regulation of apoptotic signaling in APL.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA